Th1 vaccination priming for active immunotherapy

ABSTRACT

The present invention includes vaccine compositions and methods for using these vaccine compositions in active immunotherapy. The vaccine compositions include allogeneic activated Th1 memory cells. The compositions can also include one or more disease-related antigens. The methods include administering the vaccine compositions to provide a Th1 footprint in normal individuals or patients susceptible to disease or having minimal residual disease.

The present application is based on and claims the benefit of U.S.provisional patent application Ser. No. 61/101,692, filed Oct. 1, 2008,the content of which is hereby incorporated by reference in itsentirety.

FIELD OF INVENTION

The present invention relates to the field of vaccines, and moreparticularly to therapeutic vaccine methods. Specifically, the inventionrelates to methods for use of pharmaceutical compositions containingallogeneic cells for priming for increased Th1 memory that can later beactivated and serve as an adjuvant for active immunotherapy of cancerand infectious diseases and other diseases of aging.

BACKGROUND OF THE INVENTION

Harnessing the power of the immune system to treat chronic infectiousdiseases or cancer is a major goal of immunotherapy. Vaccination (a/k/a,active immunotherapy) methods are designed to activate the immune systemto specifically recognize and protect against invading pathogens. Forover 200 years, active immunotherapy approaches have been used toprevent numerous infectious diseases, including small pox, rabies,typhoid, cholera, plague, measles, varicella, mumps, poliomyelitis,hepatitis B and the tetanus and diphtheria toxins.

Active immunotherapy concepts are now being applied to developtherapeutic cancer vaccines with the intention of treating existingtumors or preventing tumor recurrence, as well as being applied to thetreatment of chronic viral infections. However, existing activeimmunotherapy technology has not been successful in protecting againstmany of the modern disease targets such as HIV/AIDS, Hepatitis C andcancer. This is in part due to the inability of current vaccinationtechnology to elicit the correct type of immune responses.

The type of immune response generated to infection or other antigenicchallenge can generally be distinguished by the subset of T helper (Th)cells involved in the response. Immune responses can be broadly dividedinto two types: Th1 and Th2. Th1 immune activation is optimized forintracellular infections such as viruses and involves the activation ofNatural Killer (NK) cells and Cytolytic T-cells (CTL) that can lyseinfected cells, whereas Th2 immune responses are optimized for humoral(antibody) responses. Th1 immune activation is the most highly desiredfor cancer therapy and Th2 immune responses are directed more at thesecretion of specific antibodies and are relatively less important fortumor therapy. Prior art vaccine compositions are specialized ineliciting Th2 or humoral immune responses, which is not effectiveagainst cancers and most viral diseases.

Cancer eradication and maintenance of remission requires Th1 immuneactivation. Therefore, one of the goals of active immunotherapy is todevelop methods which are capable of deviating a resident Th2 responseto a Th1 response. However, in some patients which develop a potentiallyeffective Th1 immune response against a tumor or are therapeuticallyimmunized to develop a Th1 immune response, the tumors still continue togrow unaffected.

This lack of efficacy in Th1 immune patients and the ineffectiveness ofnative immune responses against tumors has been attributed to theability of tumors to employ various strategies for evasion from immuneattack. These immunoavoidance mechanisms employed by tumors render theimmune system tolerant and permit tumors to grow unimpeded by immunesurveillance even after specific upregulation of anti-tumor effectormechanisms by active immunotherapy. Therefore, active immunotherapystrategies require in addition to an immunomodulatory mechanism ofaction, a strategy to overcome tumor immunoavoidance mechanisms.

Establishment of self-tolerance to a tumor is thought to be related toexisting natural immune mechanisms which are normally employed toprevent autoimmune disease. That this normally beneficial effect may beresponsible for tumor immune evasion is supported by the observationthat many of the tolerance mechanisms that prevent autoimmunity are thesame as employed by tumors to prevent immune destruction. The “dangerhypothesis” proposes that the immune system does not primarilydiscriminate self from non-self, but instead is mainly adapted torecognize and respond to antigens depending on the context in which theantigens are presented to the immune system.

The use of adjuvants has long been a strategy for influencing the immuneresponse to antigens in a vaccine composition. Aluminum salts, andsqualene oil in water emulsion (MF59) are the most widely used adjuvantsin human vaccines. However, these adjuvants predominately promote Th2responses to antigens, and while effective at elevating serum antibodytiters do not elicit significant cellular immune responses.

SUMMARY OF THE INVENTION

This disclosure describes compositions and methods for primingindividuals with alloantigens to develop high titers of anti-allogeneicmemory cells of the Th1 phenotype. Primed individuals present with aninfectious disease or cancer can then undergo active immunotherapy withthe same alloantigens as adjuvant. The introduction of alloantigen in aprimed individual provides a strong burst of Th1 cytokines which has apowerful adjuvant effect The priming method is particularly beneficialfor elderly individuals in maintaining immune health, as the number ofTh1 cells tend to be lower as individuals increase in age.

The methods described herein provide a means to increase the number ofTh1 cells in circulation (the “Th1 footprint”) in patients by theadministration of living allogeneic cells which produce Th1 cytokines.Allogeneic cells contain alloantigens which are processed by immunecells and lead to the development of anti-allogeneic immunity. When theallogeneic cells are made to produce Th1 cytokines these cytokines serveas an adjuvant to cause the immune response to the alloantigens to besteered to Th1, thus increasing the Th1 footprint of a patient. Multipleinjections of the allogeneic cells producing Th1 cytokines serves toboost this Th1 footprint

The priming vaccinations are generally administered to patients withhigh susceptibility to diseases such as elderly patients. The primingvaccinations may also be administered to patients who have had cancerbut are in remission and might be harboring minimal residual disease butnot a full tumor challenge. The Th1 footprint generated by the primingenables the patient's immune system to be activated to produceTh1-steering cytokines at any time in the future by injecting additionalantigenic challenge. In other words, the patient's immune system isprimed and in a standby mode. Thus, when a primed patient developsdisease, vaccine compositions that include the allogeneic cells can beadministered to patients alone or in combination with antigen from thedisease. This priming of a patient to generate a Th1 footprint flowed byactive immunotherapy allows the immune system to more effectively mounta therapeutic response. Patients with existing disease can also beprimed prior to active immunotherapy.

In a one aspect, this disclosure includes a method of vaccinating apatient against a disease. The method includes administering a primingcomposition of allogeneic activated Th1 memory cells to provide a Th1footprint in the patient wherein the priming composition is administeredwhen the patient is not exhibiting symptoms of the disease.

In another aspect, this disclosure includes a method of reducingrecurrence of a disease in a patient. The method includes administeringto the patient a priming composition of allogeneic activated Th1 memorycells and disease-related antigens.

In a further aspect, this disclosure includes a method of developing aTh1 footprint in a patient to serve as an adjuvant to activateimmunotherapy. The method includes administering a priming compositionthat includes allogeneic activated Th1 memory cells wherein the primingcomposition is administered when the patient is not exhibiting symptomsof the disease.

In a yet another aspect, this disclosure includes a therapeuticcomposition for treating a disease in a patient. The compositionincludes allogeneic activated Th1 memory cells wherein the therapeuticcomposition upon administration increases the number of Th1 cells incirculation in a patient.

In yet another aspect, this disclosure includes a use of a compositioncomprising allogeneic Th1 memory cells in the manufacture of amedicament for the treatment of a disease by increasing the number ofTh1 cells in circulation after treatment.

In yet another aspect, this disclosure includes a vaccine compositionkit for a patient against a tumor or a pathogen. The kit includes apriming composition and an activating composition, wherein the primingand the activating composition both comprise allogeneic activated Th1memory cells from the same source and wherein the priming compositionupon administration increases the number of Th1 cells in circulation ina patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph showing the immune response to BCL1 without anadjuvant.

FIG. 1B is a bar graph showing the immune response to BCL1 with anadjuvant.

FIG. 2A shows plots of the phenotypic shift in CD45RB, CD62L and CD44from Day 0 to Day 6.

FIG. 2B shows plots of the phenotypic changes in the CD40L effectormolecule expression in CD4+ cells placed in culture on Day 0, Day 6 andactivated.

FIG. 2C shows plots of the phenotypic changes in the CD25 expression inCD4+ cells placed in culture on Day 0, Day 6 and activated.

FIG. 3A is a bar graph of the cytokine production of CD3/CD28cross-linked Th1 memory cells.

FIG. 3B is a bar graph of the immunogenicity of CD3/CD28 cross-linkedTh1 memory cells.

FIG. 4 is a plot showing the protective vaccination capabilities ofallogeneic activated Th1 memory cells and response to tumor challenge.

FIG. 5 is a plot showing the therapeutic vaccination capabilities ofallogeneic activated Th1 memory cells.

FIG. 6 is a plot showing the results from priming and a therapeuticbooster.

FIG. 7A is plot showing the response in a cryoablation systemic tumormodel.

FIG. 7B is a plot showing the response in a cryoablation solid tumormodel.

FIG. 7C is a plot showing the response in a solid tumor contralateral(right) tumor growth.

FIG. 7D is a plot showing systemic tumor model survival.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

The present invention includes priming vaccine compositions and methodsfor using these priming vaccine compositions in active immunotherapy.Preferably, the priming vaccine compositions include allogeneic Th1memory cells that are activated at the time of injection. The methodsinclude administering the priming vaccine compositions to provide a Th1footprint in normal individuals or patients susceptible to disease orhaving minimal residual disease. The Th1 footprint in these individualscan then be mobilized by active immunotherapy at the onset or recurrenceof a disease by administering an activating vaccine composition thatincludes allogeneic Th1 memory cells. The activating vaccine compositionmay also include disease-related antigens. The activating vaccinecomposition may be used to elicit therapeutic Th1 immunity in patientswhile also providing the means to overcome the immunoavoidancemechanisms of the disease pathogens and tumors. The priming of thepatient advantageously creates a powerful in-situ adjuvant for steeringTh1 immunity. Thus, when the patient is exposed to a pathogen or cancerchallenge the immune system is able to mount a more timely and effectiveresponse.

The present invention includes a priming vaccine composition thatcomprises living immune cells, where at least a portion are T-cells. TheT-cells are preferably memory T-cells (CD45RO+, CD62L^(Lo)) of the Th1phenotype (CD4+ T-cells that produce IFN-γ and not IL-4) and referred toherein as “memory cells” or “memory Th1 cells”. The memory Th1 cells areactivated at the time of formulation and introduction to a patient. Thepreferred activation method is by the cross-linking of CD3 and CD28surface molecules on the T-cells. The activated memory T-cellspreferably express CD40L upon being activated and produce large amountsof inflammatory cytokines (such as IFN-γ, GM-CSF, and TNF-α). Theseactivated Th1 memory cells are preferably allogeneic to the patient. Insome embodiments, the priming composition may also include diseaserelated antigenic material.

The composition with activated Th1 memory cells may be used forprophylactic purposes or therapeutic purposes, or both. The primingcompositions described herein are particularly preferable whenadministered to individuals who have not exhibited any symptoms of adisease. Even more preferable is administering the priming compositionsto patients who have not exhibited any symptoms but are susceptible to adisease. The priming composition may also be administered to patients inearly stages of a disease or in the case of cancer patients, a patientin remission or with minimal residual disease. Application of thecompositions to patients exhibiting symptoms are also within the scopeof the invention. The composition may be administered via all the routesconventionally used or recommended for vaccines: including theparenteral, intradermal, intramuscular, subcutaneous or mucosal routes.In addition, the composition may be administered intraosseally,intrathecally, interperitoneally or intralaminally. In certainembodiments, the composition may also be administered intranodally orintratumorally.

The pharmaceutical priming vaccination composition of the presentinvention includes living, activated immune cells whereby at least aportion are T-cells. The composition may also include disease-relatedantigens. Disease-related antigens as referred to herein includeantigens related to a disease causing pathogen, cells or lysates from atumor or other antigens related to a disease. The activated Th1 memorycells used in the priming vaccine pharmaceutical compositions of thepresent invention are preferably derived from normal donor blood.Preferred methods for processing and production of cells suitable foruse in the present invention are described by Har-Noy in U.S. Pat. Nos.7,435,592 and 7,402,431 and pending US published patent application2005/0191291 which are herein incorporated by reference in theirentirety.

The number of Th1 memory cells used in the vaccine compositions can varyand may also depend on the route of administration. The priming and theactivating composition generally have between about 1×10⁶ cells andabout 1×10¹⁰ cells. Preferably, the compositions have between about1×10⁶ cells and about 1×10⁸ cells. When the composition is administeredintradermally or intratumorally, between about 1×10⁷ cells and about1×10⁹ cells when the composition is administered interperitoneally,intraosseally or intrathecally and between about 1×10⁸ cells and about1×10¹⁰ cells when the composition is administered intravenously.Compositions with Th1 memory cells outside of these ranges are alsowithin the scope of this invention.

Patients that have been primed with a composition containing allogeneicTh1 memory cells will generally develop anti-alloantigen immunity.Subsequent injections of allogeneic cells can activate the pool ofanti-alloantigen cells which can release the inflammatory cytokinesnecessary for disabling immune avoidance mechanisms.

In some embodiments, the priming vaccine composition can be administeredto patients susceptible to diseases. These diseases may be diseases thatoccur often in elderly patients. The priming vaccine composition may beeffective for patients susceptible to cancer or infectious diseases suchas HIV/AIDS, Hepatitis B, Hepatitis C, herpes, tuberculosis and malaria.When the priming vaccine composition is administered to these patients,they develop an anti-alloantigen immunity. The patients' immune systemcan harbor a Th1 footprint that can be activated when necessary. Inother words, the patients' immune system is in a standby mode and primedfor a more effective Th1 response to an antigenic challenge. When thepatient develops symptoms of a particular disease, activating vaccinecompositions as described below can be administered.

In some embodiments, the priming vaccination can be administered topatients who have already had a disease such as cancer but are inremission. Patients in remission can sometimes have minimal residualdisease (MRD). In MRD, patients can still have cancer cells but inamounts that are not clinically symptomatic. Overtime, the cancer cellscan grow and lead to recurrence of the disease. Priming vaccinationsthat include the activated Th1 memory cells and one or more antigensfrom the initial cancerous cells can be administered to patients inremission to create a Th1 footprint. When the patient shows signs of arecurrence, activating vaccine compositions can be administeredcontaining the activated Th1 memory cells and one or more of thedisease-related antigens. Since the patient had been primed earlier, theresident anti-alloantigen memory can mount a response quickly to moreeffectively combat the full blown antigenic challenge.

The priming vaccine composition according to the present invention whenadministered to a patient in remission may be a composition intended forimmunization against a single pathogen or cancer, i.e. it comprises oneor more antigens from a single pathogen or cancer, or else it may be acomposition intended for immunization against several differentpathogens or cancers (this is then referred to as a vaccinecombination).

The adjuvant action of the activated Th1 memory cells can be obtainedwhen the activated Th1 cells and the disease-related antigens arecombined prior to administration. Alternatively, the activated Th1memory cells are administered separately form the disease-relatedantigen(s). Preferably, the activated Th1 memory cells are combined withthe disease-related antigens prior to administration into the patient.

In order to maintain an inflammatory environment that is capable ofdisabling the ability of tumors and disease organisms to evade immunedestruction, additional booster compositions of activated Th1 memorycells alone or formulated with antigen can be administered. Preferablythe booster compositions can be made at least 3-7 days apart, and morepreferably 7-14 days apart. Additional booster compositions can beadministered to the patient as required on a monthly or yearly basis.

The antigen component of the pharmaceutical compositions includes one ormore disease-related antigens. Any antigen source can be used in theformulation, for example these antigens can be sourced from living cellsor organisms, the source material can be irradiation inactivated (orother inactivation method), used as whole cells or organisms or lysatestherefrom. In particular, tumor cells or tumor cell lysates can serve asthe cell source material for the antigens. The cell source material canbe derived from autologous or allogeneic cell sources or from celllines. Antigen sources are described in U.S. patent application Ser. No.12/434,168 filed on May 1, 2009, incorporated herein by reference.

When the patient develops symptoms of a particular disease, activatingvaccine compositions with activated Th1 memory cells and one or moresources of disease-related antigens are administered to the patient. Thepatient's immune system can be activated to produce a full scale Th1response quickly and thus, a more effective response mounted against theantigens due to the anti-alloantigen immunity already present in thepatient.

Activating vaccine compositions generally include allogeneic activatedTh1 memory cells as described herein for the priming vaccinecompositions. Preferably, the allogeneic Th1 memory cells administeredto a patient in the activating composition are from the same source asthe allogeneic Th1 memory cells used in the priming composition. Thenumber of allogeneic activated Th1 memory cells administered to apatient can be about the same as the number of allogeneic cellsadministered in the priming compositions. Although the use of greater orlesser amounts of cells in the activating compositions are within thescope of the invention. Activating vaccine compositions may also includedisease-related antigens as described herein. The allogeneic Th1 cellsand the disease-related antigens maybe administered together as oneactivating vaccine composition. Alternatively, the allogeneic Th1 cellsand the disease-related antigens can be administered as separatecompositions.

EXAMPLE

Mice—Five to six week old female Balb/c (H-2^(d/d)) and male C57BL/6(H-2^(b/b)) mice were purchased from the Hebrew University-HadassahMedical School Animal Facility, Jerusalem, Israel. All mice were keptunder specific pathogen-free (SPF) conditions and given acidified waterand food ad libitum. The study was approved by the Animal EthicalCommittee of the Hebrew University Medical School. All mice were 6 to 8weeks old when placed on experiment.

BCL1 Tumor Model—BCL1 is a spontaneous B-cell leukemia/lymphoma ofBalb/c origin. The BCL1 tumor line is maintained in viva by serialpassages in Balb/c recipients. In these experiments, animals wereinfused intravenously through the tail vein with 2000 BCL1 cells on day0 which is lethal in 100% of the mice. In some experiments, 1×10⁴ BCL1were implanted subcutaneously in the flank of Balb/c mice. BCL1 forms arapidly growing solid tumor in this setting resemblinglymphoma/plasmacytoma.

Preparation of Allogeneic Th1 Memory Cells—Allogeneic Th1 memory cellswere prepared. Briefly, CD4+ cells were isolated from male C57BL/6spleens and cultured for 6 days with anti-CD3 and anti-CD28-coatedparamagnetic beads (CD3/CD28 T-cell Expander beads, Dynal/Invitrogen) atan initial bead:CD4 cell ratio of 3:1 and 20 IU/mL recombinant mouse(rm)IL-2, 20 ng/mL rmIL-7, 10 ng/mL rmIL-12 (Peprotech, N.J.) and 10μg/mL antimurine IL-4 mAb (Becton Dickenson) in RPMI 1640 mediacontaining 10% FBS, penicillin-streptomycin-glutamine, nonessentialamino acids (NEAA) (Biological Industries, Israel) and 3.3 mMN-acetyl-cysteine (NAC; Sigma) (complete media). After 6 days inculture, the CD4 cells were harvested and debeaded by physicaldisruption and passage over a magnet. These cells were either used freshor stored in liquid nitrogen for future use. Prior to use, the cellswere activated by incubation with anti-CD3/anti-CD28-coated nanobeadsfor 4 h in complete media.

Vaccinations—Mice were vaccinated with vaccine compositions suspended in0.1 ml of HBSS or complete media. Inoculations were made either inalternating foot pads or in the skin layer of the shaved flank.

BCL1 tumor antigen preparations—BCL1 lysates and irradiated BCL1 cellswere used as sources of tumor antigens. Batches of BCL1 lysate werederived from 1×10⁷ BCL1 cells suspended in 2 mL of HBSS and lysed by 3freeze (in −80° C. freezer)-thaw (37° C. water bath) cycles. Total celldisruption was microscopically validated using trypan blue staining. Thelysate was mixed well to assure a homogenous solution and aliquoted intoseparate 0.2 ml doses. These doses were stored at −80° C. prior to use.Fresh BCL1 cells were irradiated at 20 Gy and used within an hour oftreatment.

Monoclonal Antibodies—The following monoclonal antibodies (mAb) wereused to surface phenotyping: anti-mCD4-PerCP-Cy5 (IgG2a); isotypecontrol rat IgG2a-PerCP-Cy5.5; anti-mCD62L-APC (IgG2a); isotype controlrat IgG2a-APC; anti-mCD45RB-PE (IgG2a); isotype control rat IgG2a-PE;anti-mCD8a-FITC (IgG2a); isotype control rat IgG2a-FITC;anti-mCD44-FITC(IgG2b); isotype Control rat IgG2b-FITC;anti-mCD154(CD40L)-PE (IgG); isotype control rat IgG-PE; anti-mCD25-APC(IgG1); isotype control rate IgG1-APC; anti-mCD3e-PerCP-Cy5.5(IgG);isotype control Armeniam hamster IgG-PERCP-Cy5.5 all from eBioscience,Inc. (San Diego, USA).

ELISPOT Assay—Single cell suspensions of spleen cells from immunizedmice were prepared. The cells were aliquoted so that 2×10⁶ viable cellswere plated in 2 ml of complete media in wells of a 24 well plate. Thesplenocytes in each well were pulsed with test antigens. The testantigens were prepared as freeze-thaw lysates suspended in completemedia. Each well was pulsed with lysate from 1×10⁷ cells: either BCL1,allogeneic Th1 cells or splenocytes from untreated syngeneic mice. Thepulsed wells were cultured for 24 h at 37° C. in a humidified CO₂incubator. After 24 h, the non-adherent cells were harvested, washed andcounted. These cells were then plated in triplicate at 100,000 viablecells per well on pre-coated anti-MN-7 and anti-IL-4 plates(eBioScience, San Diego, Calif.) and incubated for an additional 20 h incomplete media supplemented with 20 IU/ml mL-2 (Peprotech). Freshsplenocytes from syngeneic mice activated with PHA were used as apositive control for each plate (data not shown). The plates weredeveloped in accordance with the manufacturer's instructions and read onan automated image analysis system.

Cryoimmunotherapy tumor models—Two tumor models were used for thecryoimmunotherapy protocols, a bilateral solid tumor model and solidtumor with systemic disease model. The bilateral solid tumor modelconsisted of mice given subcutaneous injections of 1×10⁴ BCL1 tumorcells bilaterally in the shaved flanks on day 0. For the solid tumorwith systemic disease model, mice received a single subcutaneousinoculation of 1×10⁴ BCL1 on day 0 on the left flank and also anintravenous infusion of 2000 BCL1.

Cryoablation—Anesthetized mice (ketamine-HCL, 100 mg/kg, i.p.) underwentcryoablation treatment by applying mild pressure for 10 seconds withfrozen tweezers (which were kept in liquid nitrogen) to the tumor.Tumors were 16-25 mm² when treated. The ice ball covered the completetumor mass. To ensure complete thawing of the treated area beforevaccination, intratumoral treatments were administered after 1 hour.

Statistics—Two-way ANOVA was used to determine significant differencesin cytokine levels, ELISPOT response frequencies and tumor volumechanges. A P value of <0.05 was considered significant. Logrank andhazard ratio analysis was used to compare Kaplan-Meier survival curves(Graphpad Software; San Diego, Calif.). Animals that survived >60 dayswere censored from the analysis.

Immune response to BCL1 vaccination with or without adjuvant. (FIG. 1aand FIG. 1b ) Balb/c mice (n=3) were vaccinated four times intradermallyat weekly intervals with either freeze-thawed BCL1 tumor lysate (f/tBCL1) or irradiated BCL1 (irrad BCL1) without adjuvant (FIG. 1A) ormixed with 1×10³ CD3/CD28 cross-linked allogeneic Th1 cells (allo Th1)as adjuvant (FIG. 1B). On the 5th week following the first inoculation,animals were sacrificed and their spleens harvested, single cellsuspension cultures of splenocytes were pulsed with f/t BCL1 or f/tsplenocytes from a naive Balb/c mouse as control. After 24 h, thenon-adherent T-cells were removed and plated in triplicates at 1×10⁵cells per well of anti-IFN-γ and anti-IL-4-coated ELISPOT plates. Aftera culture of 20 h, IFN-γ and IL-4 spots were developed and counted bycomputer-assisted video image analysis. Each bar represents the meanspot number of triplicates +/− SE out of 10⁵ T-cells. Asterisk (*) onFIG. 1A and FIG. 1B indicates significant difference (p<0.05) and n.s.indicates not significant (p>0.05) compared to control and betweenbracketed values (ANOVA 2-tailed test).

Characterization of CD3/CD28 cross-linked Th1 cells. (FIG. 2A, FIG. 2Band FIG. 2C) C57BL/6-derived positively selected CD4+ T-cells wereplaced in culture on day 0 in cRPMI supplemented with rmIL-12, rmIL-7,rmIL-2 and a neutralizing anti-IL-4 mAb and activated with CD3/CD28conjugated T-Cell Expanded beads (Dynal/Invirogen) at a 3:1 bead:cellratio. The cells were split daily from day 3-6 and supplemented withadditional T-cell Expander beads, rmIL-7, rmIL-2 and anti-IL-4 mAb. Onday 6, the cells were harvested and activated with CD3/CD28 conjugatednanobeads. The phenotypic shift in CD45RB, CD62L and CD44 from Day 0 toDay 6 is shown in FIG. 2A. The black filled area represents the isotypecontrol. The black line is the phenotype of Day 0 CD4+ cells. The grayfilled area is the phenotype of day 6 cells prior to CD3/CD28 nanobeadactivation and the gray line represents the phenotype after CD3/CD28nanobead activation. FIG. 2B shows the phenotypic changes in the CD40Leffector molecule expression in CD4+ cells placed in culture on day 0compared to Day 6 harvested cells before and after CD3/CD28 nanobeadactivation. Only the activated cells expressed significant amounts ofthis effector molecule. FIG. 2C represents the phenotypic change in CD25expression in Day 0 CD4+ cells compared to Day 6 cells before and afterCD3/CD28 nanobead activation. The phenotype of the activated day 6 cellswas CD4+, CD62Llo, CD45RBhi, CD44hi, CD40L+, CD25+.

Cytokine production (FIG. 3A) and immunogenicity (FIG. 3B) of CD3/CD28cross-linked Th1 memory cells. IFN-□ and IL-4 cytokine production wasanalyzed in supernatants from 6 h cell cultures by ELISA. CD4 cellspositively selected from C57BL/6 cultured for 6 days in the presence ofIL-12 (day 1-3 only), IL-7 and IL-2 expanded 60-100-fold anddifferentiated to CD45RBhi, CD44hi effector/memory cells. These cellswere harvested on Day 6, washed and reactivated with CD3/CD28 nanobeads,cultured for 6 h and the supernatants collected for analysis of by ELISA(FIG. 3A: Fresh activated Th1). These results were compared tosupernatants from 6 h cultures of the same Day 6 harvested cells thatwere first frozen in liquid nitrogen and then later thawed and activatedwith CD3/CD28 nanobeads (FIG. 3A: thawed activated Th1). For comparison,supernatants from a sample of positively selected CD4 cells (the samethat were placed in culture on Day 0) were activated with CD3/CD28T-cell Expander Beads at a 3:1 bead:cell ratio and cultured for 6 h(FIG. 3A: CD4 naïve). Supernatants from Day 6 harvested cells were alsocultured for 6 h without CD3/CD28 activation (FIG. 3A: nonactivated Th1fresh).

The immunogenicity of these cell compositions were tested by ELISPOT(FIG. 3B). 1×10⁴ fresh activated Th1 cells, fresh non-activated Th1cells or positively selected CD4 cells (control) derived from C57BL/6mice were inoculated intradermally in allogeneic Balb/c mice once a weekfor 4 weeks. During the 5th week, the mice (n=3) were sacrificed,spleens removed and

single cell splenocytes cultures established. The cultures were pulsedwith freeze/thawed lysates of CD57BL/6 splenocytes and cultured for 24h. The non-adherent T-cell fraction was then harvested, washed, and1×10⁵ cells were transferred to anti-IFN-γ or anti-IL-4 coated ELISPOTplates in triplicate and cultured another 20 h in the presence of 20 IUrmIL-2. Each bar represents the mean spot number of triplicates ±SE.

Protective vaccination and tumor challenge. Kaplan-Meier survival curvesof Balb/c mice vaccinated against BCL1 tumor (n=8 for each group). (FIG.4) All mice were infused i.v. with 2000 cells of BCL1 on day 0. Prior totumor challenge, mice were inoculated i.d. on day −22, day −15, day −8and day −1 with either media alone (control), 1×10⁴ allogeneic CD3/CD28cross-linked Th1 cells (Th1 alone), 1×10⁴ allogeneic CD3/CD28cross-linked Th1 cells mixed with irradiated BCL1 (irrad BCL1+Th1) or1×10⁴ allogeneic CD3/CD28 cross-linked Th1 cells mixed withfreeze/thawed BCL1 tumor lysate (f/t BCL1+Th1). The median survival ofthe control mice was 21 days. Vaccination with irrad BCL1 alone (mediansurvival=20.5 days) or f/t BCL1 alone (median survival=20.0 days) didnot significantly affect survival compared to control. The Th1 alonepretreatment significantly extended survival to a mean of 24 days(hazard ratio-3.14). Vaccination with irrad BCL1 with Th1 as an adjuvantdid not have a significant effect on survival (mean survival=22 days).Mixing f/t BCL1 with Th1 resulted in 50% of the mice surviving lethaltumor challenge and resulted in a median survival of 46 days (hazardratio=6.08).

Therapeutic vaccination. Kaplan-Meier survival curves of Balb/c mice

(n=6 per group) infused with 2000 BCL1 iv on day 0. (FIG. 5) Mice weregiven id injections of either 1×10⁴ allogeneic CD3/CD28 cross-linked Th1memory cells alone (Th1) or f/t lysate of 1×10⁶ BCL1 mixed with Th1 (f/tBCL1+Th1) i.d. on days 1, 8 and 15. Control mice survived a mean of 19.5days. Mice vaccinated with Th1 alone survived significantly longer thancontrol with a mean of 26 days (logrank: p=0.001; hazard ratio: 3.981).Mice vaccinated with f/t BCL1+Th1 also survived significantly longerthan control mice with a mean survival of 34 days (logrank: p=0.001;hazard ratio: 3.981), which was not significantly different than the Th1alone group.

Prime with therapeutic booster. Kaplan-Meier survival curves of Balb/cmice vaccinated against BCL1 tumor (n=8 for each group). (FIG. 6) Allmice were infused iv with 2000 cells of BCL1 on day 0. Prior to tumorchallenge, mice were primed by inoculation i.d. on day −22, day 15, day−8 and day −1 with either media alone (control), 1×10⁴ allogeneicCD3/CD28 cross-linked Th1 cells (Th1) or 1×10⁴ Th1 mixed withfreeze/thawed BCL1 tumor lysate (Th1+f/t BCL1 prime). On day 7, somemice were administered therapeutic booster inoculations i.d. with eitherTh1 alone or Th1 mixed with f/t BCL1. Control mice survived a median of20 days. Vaccination with Th1 alone resulted in significant survivalextension to a median of 23 days (logrank: p=0.011;

hazard ratio: 2.478). Vaccination with Th1 alone followed by a Th1booster resulted in significant survival advantage (median survival=25days) compared to control (logrank: p<0.0001; hazard ratio-3.915) andsignificant survival compared to Th1 alone (logrank: p=0.03; hazardratio=2.337). Mice primed by vaccination with f/t BCL1 with Th1 asadjuvant (Th1+f/t BCL1 prime) resulted in median survival of 57.5 dayswith 50% of mice surviving lethal challenge. Mice primed with Th1+f/tBCL1 and administered a Th1 booster had 75% survival lethal challenge.Mice primed with Th1 cells alone and administered a therapeutic boosterinjection with f/t BCL1+Th1 had a significant survival

advantage compared to control with median survival of 46 days (logrank:p<0.0001; hazard ratio=5.633) and 37.5% of mice surviving lethalchallenge.

Solid tumor and systemic tumor response to cryoimmunotherapy. In thesolid tumor model, Balb/c mice were administered subcutaneousinoculations of 1×10⁴ BCL1 tumor cells bilaterally on the shaved flankson day 0. In the systemic tumor model, Balb/c mice were administeredinoculations of 1×10⁴ BCL1 tumor cells on the left flank and 2000 BCL1intravenously through the tail vein on day 0. The Kaplan-Meier survivalcurves of systemic tumor model (FIG. 7A) and solid tumor model (FIG. 7B)mice (n=8 each group) treated on day 14 with cryoablation of allobservable tumor of the left tumor mass either alone (cryo alone) orwith intratumoral allogeneic CD3/CD28 cross-linked Th1 memory cells(Th1) (cryo+Th1) or with intratumoral Th1 cells alone withoutcryoablation (Th1 alone). Control mice survived a median of 21 days inthe systemic model and 28.5 days in the solid tumor model. The cryo+Th1treatment resulted in significant survival advantage (logrank: p=0.0059;hazard ratio=3.194) in the systemic model. The survival advantage in thesolid tumor model was not significant (n.s.). The same experiment wasrepeated with the addition of a 1×10⁵ intravenous infusion of Th1 on day7 for all mice. FIG. 7C shows the tumor growth curves of thecontralateral untreated tumor masses for mice treated with thisprotocol. 40% of the mice treated with cryo+Th1 were cured of disease.The growth of contralateral tumor was significantly suppressed (p<0.01)in the other 60% of mice that eventually succumbed to disease. In thesystemic tumor model (FIG. 7D), Kaplan-Meier survival curves are shown.Mice treated with intratumoral Th1 cells alone survived a mean of 28days, which was significantly longer than control mice which survived amean of 19 days (logrank: p<0.0001; hazard ratio=4.291). 40% of micetreated with cryo+Th1 survived >90 days.

Results

BCL1 Immunogenicity—The native immune response to BCL1 vaccination inBalb/c mice was characterized to serve as a baseline in which to analyzethe biological effects of adding an adjuvant. Since BCL1 has beencontinuously passaged in-vivo for many years that the current BCL1 clonewould prove to be non-immunogenic when administered to syngeneic Balb/cmice without an adjuvant due to “immunoediting”.

The immunogenicity of two vaccine preparations of BCL1, eitherirradiated whole BCL1 (irrad BCL1) or a freeze-thawed lysate of BCL1(f/t BCL1) were tested without adjuvant (FIG. 1A) or mixed with 1×10³CD3/CD28 cross-linked allogeneic Th1 cells (allo Th1) as adjuvant (FIG.1B). The “danger hypothesis” predicts that the f/t BCL1 lysatepreparation would be more immunogenic than the irrad BCL1 preparation.To test the immunogenicity of f/t BCL1 and irrad BCL1 preparations theywere each administered intradermally (i.d.) to Balb/c mice in 0.2 ml ofHBSS once a week for 4 weeks.

Vaccination with both irrad BCL1 and f/t BCL1 without adjuvant were ableto elicit significant tumor-specific immune responses (see FIG. 1A). Themean frequency of tumor-specific T-cells (IL-4+IFN-γ spots) was 1/64after vaccination with irrad BCL1, which was significantly greater(3.5-fold) than the mean frequency of 1/227 after f/t BCL1 vaccination.Both vaccination protocols resulted in significantly greater frequenciesof responding T-cells compared to the control mean frequency of 1/3333(p<0.001).

Both vaccine preparations caused tumor-specific T-cell responses biasedto Th2 (IL-4). T-cell IL-4 mean frequency response to irrad BCL1vaccination was 1/93 and the IFN-γ response was significantly lower(p<0.001) at 1/208. The mean frequency of IL-4 responders in f/t BCL1vaccinated mice was 1/322 which was significantly higher (p<0.01) thanthe IFN-γ mean response frequency of 1/769. The mean frequency of IFN-γresponders in irrad BCL1 vaccinated mice compared to f/t BCL1 vaccinatedmice were not significantly different.

None of the mice vaccinated with irrad BCL1 or f/t BCL1 were able tosurvive a lethal challenge of 2000 BCL1 cells administered intravenouslythrough the tail vein (See FIG. 4), indicating the significant immuneresponses to both BCL1 vaccination protocols were not protective.

Characterization of CD3/CD28 cross-linked Th1 Memory cells—TheC57BL/6-derived CD3/CD28 cross-linked Th1 memory cells were firstcharacterized for the surface phenotype (FIG. 2A-C), cytokine productionprofile (FIG. 3A) and immune response after 4 weekly i.d. injections inallogeneic Balb/c mice (FIG. 3B).

The surface expression of CD62L, CD45RB, CD44, CD25 and CD40L wasanalyzed to characterize the differentiation of cells over the 6 dayculture by FACS of day 0 CD4+ source cells and compared the stainingpatterns to the same cells harvested after 6 days in culture before andafter activation by CD3/CD28 cross-linking. Results are shown in FIG.2A.

The day 0 positively selected CD4+ cells from splenocytes were stainedCD62L^(hi), CD44^(hi) but converted from a CD62L^(hi) phenotype to aCD62L^(lo) phenotype. Therefore, the day 6 CD3/CD28 cross-linked cellsexpressed a phenotype of CD4+, CD62L^(lo), CD45RB^(hi), CD44^(hi).

Phenotypically, mouse memory cells are normally CD62L^(lo), CD45RB^(lo),CD44^(hi). The CD62L^(lo), CD45RB^(lo), CD44^(hi) phenotype of cellsproduced by our culture method express the same phenotype asmemory/effector cells that have been previously associated withautoimmune disease and allograft rejection.

Activation by CD3/CD28 cross-linking of day 6 cells for 4 h caused asignificant increase in the number of cells expressing CD40L from 9.27%to 67.65% (FIG. 2B) which correlated with an increase in cellsexpressing CD25 from 63.09% to 91.52% (FIG. 2C).

These CD3/CD28-activated CD4⁺, CD62L^(lo), CD45RB^(hi), CD44^(hi),CD40L⁺, CD40L⁺, CD25⁺ memory/effector cells (n=6 batches) were testedfor cytokine production after activation by CD3/CD28 cross-linking offresh day 6 harvested cells or day 6 harvested cells that had beenfrozen in liquid nitrogen, thawed and activated. Activated Day 0 CD4+cells and non-activated fresh harvested Day 6 cells were included forcomparison (see FIG. 3).

Fresh activated CD4+ memory/effector cells expressed substantial amountsof IFN-γ (4210±169.7 pg/ml/6 h) and negligible IL-4 (52.33±6.8 pg/ml/6h) and thus these cells are referred to as Th1 memory cells. When theseTh1 memory cells are frozen in liquid nitrogen prior to activation andlater thawed and activated, they maintain the Th1 phenotype but expressapproximately 29% less IFN-γ and IL-4 than the fresh cells (2985±173.5pg/ml/6 h of IFN-γ and 37.2±6.95 pg/ml/6 h of IL-4). Non-activated Th1memory cells produced negligible amounts of cytokines (13±6.7 pg/ml/6 hIFN-γ, 8.8±3.6 pg/ml/6 h IL-4). The source CD4+ cells isolated bypositive selection from a single cell suspension of normal C57BL/6splenocytes produced cytokines upon activation with CD3/CD28-conjugatedmicrobeads with a Th2 bias (254±50.2 pg/ml/6 h IL-4; 53.5±11.4 pg/ml/6 hIFN-γ).

These data demonstrate that the culture conditions cause CD4+ naïvecells with a Th2 bias to differentiate to strongly polarized Th1memory/effector cells which express CD40L upon CD3/CD28 cross-linkingand express an unusual CD62L^(lo), CD45RB^(hi), CD44^(hi) memoryphenotype.

Immune Response to Allogeneic CD3/CD28 Cross-Linked Th1 Memory Cells—Tocharacterize the potential of allogeneic CD3/CD28 cross-linked Th1memory cells to provide adjuvant activity for promotion of Type 1immunity, a study was conducted to determine if the C57BL/6 derived Th1memory cells were able to elicit Type 1 immunity to their ownalloantigens in allogeneic Balb/c hosts. Mice were administered 1×10⁴CD3/CD28 cross-linked Th1 memory cells or 1×10⁴ Th1 memory cells withoutCD3/CD28 cross-linking i.d. in 0.1 ml of cRPMI, inoculation of cRPMIalong was used as a control. Mice (n=6) were inoculated once weekly forfour weeks.

During the 5^(th) week after the first vaccination, the mice weresacrificed and spleens were removed and single cell suspensions wereprepared for ELISPOT analysis as previously. Results are shown in FIG.3B.

Vaccination with allogeneic CD4 cells (Day 0) alone resulted in a baseline allogeneic immune response means frequency (IFN-γ+IL-4) of 1/348T-cells. Allogeneic Th1 cells (Day 6) without CD3/CD28 cross-linkingresulted in a response mean frequency of 1/268 T-cells, which was notsignificantly different than the CD4 cells alone. However, CD3/CD28cross-linking of the allogeneic Th1 cells resulted in a mean frequencyof 1/34 alloantigen-specific T-cells, which was significantly greaterthan the response frequency of allogeneic CD4 cells and non-activatedallogeneic Th1 cells (p<0.0001).

The immune response to allogeneic CD4 cells and non-activated allogeneicTh1 memory cells resulted in skewed Th2 immunity, while the CD3/CD28cross-linked allogeneic Th1 memory cells elicited a strongly polarizedTh1 response. CD4 cell vaccination resulted in a mean frequency of 1/181IL-4 producing T-cells, which was significantly greater (p<0.0001) thanthe mean frequency of 1/4167 IFN-γ producing T-cells. Non-activatedallogeneic Th1 memory cells elicited a mean frequency of 1/147 IL-4responding T-cells, which was significantly greater (p<0.0001) than themean frequency of 1/1587 IFN-γ responding T-cells. The syngeneicsplenocyte control did not elicit a detectable adaptive immune response(results not shown).

Protective Vaccination and Challenge—In order to determine if allogeneicTh1 cells could serve as an adjuvant to protect mice from a lethalchallenge of BCL1 tumor, vaccine mixtures were prepared of eitherfreeze/thawed BCL2 lysate (f/t BCL1) or irradiated BCL1 cells (irradBCL1) used alone (control) or mixed with 1×10⁴ allogeneic Th1 cells.Allogeneic Th1 cells alone without a source of tumor antigen were usedas a control. Mice (n=8 in each group) received 4 i.d. inoculations ofthe vaccine preparations at weekly intervals on days −22, −15, −8 and−1. Media alone injections were used as a control. On day 0, allvaccinated mice received a lethal i.v. challenge of 2000 BCL1 cells.

Mice were followed for survival (see FIG. 4). The median survival of themedia alone control mice was 21 days. Vaccination with either irradBCL1, f/t BCL1 or allogeneic Th1 alone did not result in protection fromtumor challenge. Interestingly, the allogeneic Th1 alone vaccination,while not protective, significantly extended survival of challenged miceto a mean of 24 days (hazard ratio-3.14). Vaccination with a compositionof irrad BCL1+TH1 cells did not affect survival (median survival-22days) and did not provide protection. Vaccination with f/t BCL1+Th1cells resulted in a median survival of 46 days (hazard ratio=6.08) with50% of the mice surviving lethal tumor challenge.

Therapeutic Vaccine Protocol—The above data demonstrated the protectiveeffects of vaccination with Ft BCL1 and allogeneic CD3/CD28 cross-linkedTh1 memory cells used as an adjuvant in animals that were vaccinatedwhen they were tumor free and then challenged with a lethal dose oftumor. In order to determine if this protective effect could alsoprovide a therapeutic effect, vaccination protocols were investigated inmice with pre-existing tumors.

The previous protective vaccination protocol included 4 weekly i.d.inoculations (28 days) followed by tumor challenge on day 35. Thisvaccination schedule could not be translated to the therapeutic settingin our model, as mice succumb to disease 19-22 days after lethal BCL1infusion. Therefore, mice (n=6 each group) received a lethal dose of2000 BCL1 cells intravenously on day 0 and therapeutic vaccinations ofeither 1×10⁴ allogeneic CD3/CD28 cross-linked Th1 memory cells alone(Th1) or f/t lysate of 1×10⁶ BCL1 mixed with allogeneic Th1 (f/tBCL1+Th1) i.d. on days 1, 8 and 15. Media alone inoculations on the sameschedule served as control. Results are shown in FIG. 5.

Control mice survived a mean of 19.5 days. Interestingly, micevaccinated with Th1 alone without any source of tumor antigen survivedsignificantly longer than control with a mean of 26 days (logrank:p=0.001; hazard ratio: 3.981). Mice vaccinated with f/t BCL1+Th1 alsosurvived significantly longer than control mice with a mean survival of34 days (logrank: p=0.001; hazard ratio: 3.981), but this was notsignificantly different than the allogeneic Th1 alone group. No micewere cured with either of these therapeutic vaccine protocols.

Prime and Therapeutic Booster Vaccination—Since no mice were cured inthe therapeutic vaccine protocols, the 19-22 day lifespan of miceinfused with BCL1 tumors was not sufficient time to develop an adoptiveimmune response that could overwhelm the rapidly growing tumor.Therefore, tests were run to determine whether mice that were primedwith allogeneic Th1-containing vaccinations prior to tumor challengewould respond better to therapeutic vaccination. In these experiments,mice were primed by i.d. inoculations on day −22, day −15, day −8 andday −1 with either media alone (control), 1×10⁴ allogeneic CD3/CD28cross-linked Th1 cells (Th1) or 1×10⁴ Th1 mixed with freeze/thawed BCL1tumor lysate (Th1+f/t BCL1 prime). All mice were infused with 2000 BCL1on day 0. On day 7, some mice were administered i.d. therapeutic boosterinoculations of either 1×10⁴ Th1 alone or 1×10⁴ Th1 mixed with f/t BCL1.Results are shown in FIG. 6.

Control mice survived a median of 20 days. Priming with Th1 alone againresulted in significant survival extension to a median of 23 days(logrank: p=0.011; hazard ratio: 2.478). Priming with Th1 alone followedby a Th1 therapeutic booster resulted in significant survival advantage(median survival=25 days) compared to control (logrank: p<0.0001; hazardratio=3.915) and significant survival compared to Th1 prime alone(logrank: p=0.03; hazard ratio=2.337). Mice primed by vaccination withf/t BCL1 with Th1 as adjuvant (Th1+f/t BCL1 prime) had median survivalof 57.5 days with 50% of mice surviving lethal challenge, the sameresult obtained in our previous experiment (see FIG. 4). Mice primedwith Th1+f/t BCL1 and administered a Th1 therapeutic booster had 75%survival after a lethal challenge. Mice primed with Th1 cells alone andadministered a therapeutic booster injection with f/t BCL1+Th1 had asignificant survival advantage compared to control with median survivalof 46 days (logrank: p<0.0001; hazard ratio+5.633) and 37.5% of micesurviving lethal challenge.

The shape of the Kaplan-Meier survival curve for the Th1 prime/Th1+f/tBCL1 booster group was different when compared to other groups that alsoresulted in mice cured after lethal BCL1 injection. The 50% of mice thatdid not survive after Th1+f/t BCL1 prime and the 25% that did notsurvive lethal challenge from the Th1+f/t BCL1 prime/Th1 booster groupshowed immediate signs of leukemia (palpable splenomegaly andsignificant weight gain) and succumbed to the disease very early at amean of 24 days. By contrast, the Th1 prime+f/t BCL1 treatment groupcontained a subset of mice (62.5%) also with obvious leukemia but thatsurvived significantly longer than control mice and a separate subset(37.5%) that were apparently cured and never showed signs of leukemia.

Cryoimmunotherapy—In order to try to further improve the efficacy oftherapeutic vaccination, it was hypothesized that in-situ tumor death bynecrosis would provide a more potent adaptive immune response than ourother freeze-thawed lysate preparations. Neurotically killed cells areknown to activate endogenous signals of distress responsible for therecruitment and maturation of DC, stimuli that would note be generatedby healthy or apoptotically dying cells and may be missing from ourlysate preparations. Further, exposure of immature DC to these stimuliprovides maturation signals, critical for the initiation of local andsystematic Th1 immunity.

In order to cause in-situ death by necrosis, a cryoablation protocol wasused. Cryoablation surgery is a technique that can be translated to theclinic and has been shown to be a well-aimed and controlled procedurecapable of inducing tissular necrosis. Cryoablation has been known toelicit an antigenic stimulus (capable of generating a specificimmunologic response against autologous antigens of the frozen tissue).

Two tumor models were established where subcutaneous tumors wereavailable for cryoblation. In the solid tumor model, Balb/c mice wereadministered subcutaneous inoculations of 1×10⁴ BCL1 tumor cellsbilaterally on the shaved flanks on day 0. In the systemic tumor model,Balb/c mice were administered inoculous of 1×10⁴ BCL1 tumor cells on theleft flank and 2000 BCL1 Intravenously through the tail vein on day 0.The left tumor of these animals (n=8 per group) were then treated on day14 after the solid tumors had grown to an area >16 mm² with cryoblationalone, cryoblation with intratumoral 1×10³ allogeneic CD3/Cd28cross-linked Th1 memory cells (Th1), Th1 cells alone or intratumoralcomplete media alone as control. The results are shown in FIGS. 7A and7B.

In the systemic tumor model (FIG. 7A), the mean survival of control micewas 21 days. Survival of mice treated with cryoblation alone and Th1alone was not different than control. However, the combination ofcryoblation with intratumoral Th1 treatment resulted in significantlyextended survival to a mean of 28.5 days (logrank: p=0.0057; hazardratio: 3.194). In the solid tumor model (FIG. 7B), the mean survival ofcontrol mice was 28 days. None of the treatments tested in this modelprovided a significant survival advantage.

Because the cryoblation protocol did not result in any mice cured asresult of the treatment, we modified the treatment to include a 1×10⁵intravenous infusion of allogeneic Th1 on day 7 for all mice. It waspreviously shown that intravenous infusion of 1×10⁵ allogeneic Th1 cellson day 7 caused a significant survival advantage in mice lethallyinjected with BCL1 on day 0. It was hypothesized that this treatmentwould provide more time for a potentially curative adaptive immuneresponse to develop and thus affect the survival of mice undergoingcryoimmunotherapy. Further, this treatment would prime for alloantigenimmunity, shown earlier (FIG. 6) to provide a survival advantage.

The results of these studies are shown in FIG. 7C (solid tumor model)and FIG. 7D (systemic tumor model). In the solid tumor model, the areaof tumor was determined by measurement of the longest width and lengthof the tumor with calipers. After complete ablation of the left tumor bycryoablation, only contralateral untreated tumor mass was measured. Thetumor growth curves of the contralateral alone and mice treated withcombination of cryoblation and intratumoral allogeneic Th1 (cryo+Th1).40% of mice treated with the combination therapy survived withoutevidence of tumor. The contralateral tumor area is displayed toseparately show the response of the 40% mice that survived and the 60%that eventually succumbed to disease. The growth of the contralateraltumors were significantly suppressed (p<0.01) in the 60% of mice thateventually succumbed to disease. In the systemic tumor model (FIG. 7D),mice treated with intratumoral allogeneic Th1 cells alone survived amean of 28 days, which was significantly longer than control mice whichsurvived a mean of 19 days (logrank: p<010001; hazard ratio=4.291). 40%of mice treated with cryo+Th1 survived >90 days.

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

What is claimed is:
 1. A method of treating an individual susceptible tocancer, the method comprising: developing an anti-alloantigen immunityin the individual prophylactically, prior to onset of the cancer byadministering to the individual a priming composition comprisingallogeneic activated Th1 memory cells and cross-linking agents forcross-linking the CD3 and CD28 surface moieties on the Th1 memory cells,wherein the Th1 memory cells are activated by the cross-linking of theCD3 and CD28 surface molecules at the time of administration, whereinadministration of the priming composition causes increased Th1anti-alloantigen specific titer in circulation, wherein theanti-alloantigen immunity is developed in the individual prior toadministration of cancer related antigens; and administering anactivating composition comprising allogeneic activated Th1 memory cellsand one or more cancer-related antigens to the primed individual,wherein the activating composition is administered after the onset ofcancer in the individual.
 2. The method of claim 1 further comprisingadministering one or more booster compositions prior to the individualdeveloping symptoms of cancer.
 3. The method of claim 2 wherein thebooster composition is administered at least about 3-7 days after theprevious administration of allogeneic cells.
 4. The method of claim 2wherein the booster composition comprises allogeneic activated Th1memory cells.
 5. The method of claim 4 wherein the booster compositionfurther comprises one or more disease-related antigens.
 6. The method ofclaim 1 wherein the individual is in remission from cancer.